Clinical Science (2001) 101, 629–635 (Printed in Great Britain)
Flow-mediated dilatation following wrist and
upper arm occlusion in humans: the
contribution of nitric oxide
Sagar N. DOSHI*, Katerina K. NAKA†, Nicola PAYNE‡, Christopher J. H. JONES†,
Moira ASHTON†, Malcolm J. LEWIS* and Jonathan GOODFELLOW†
*Department of Pharmacology, Cardiovascular Sciences Research Group, Wales Heart Research Institute, University of Wales
College of Medicine, Heath Park, Cardiff CF14 4XN, U.K., †Department of Cardiology, Cardiovascular Sciences Research Group,
Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K., and ‡Department
of Medical Computing and Statistics, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K.
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Flow-mediated dilatation (FMD) of the brachial artery assessed by high-resolution ultrasound is
widely used to measure endothelial function. However, the technique is not standardized, with
different groups using occlusion of either the wrist or the upper arm to induce increased blood
flow. The validity of the test as a marker of endothelial function rests on the assumption that the
dilatation observed is endothelium-dependent and mediated by nitric oxide (NO). We sought to
compare the NO component of brachial artery dilatation observed following wrist or upper arm
occlusion. Dilatation was assessed before and during intra-arterial infusion of the NO synthase
inhibitor N G-monomethyl-L-arginine (L-NMMA) following occlusion of (i) the wrist (distal to
ultrasound probe) and (ii) the upper arm (proximal to ultrasound probe) for 5 min in ten healthy
males. Dilatation was significantly greater after upper arm occlusion (upper arm, 11.62p3.17 % ;
wrist, 7.25p2.49 % ; P l 0.003). During L-NMMA infusion, dilatation after wrist occlusion was
abolished (from 7.25p2.49 % to 0.16p2.24 % ; P 0.001), whereas dilatation after upper arm
occlusion was only partially attenuated (from 11.62p3.17 % to 7.51p2.34 % ; P l 0.006). The peak
flow stimulus was similar after wrist and upper arm occlusion. We conclude that dilatation
following upper arm occlusion is greater than that observed after wrist occlusion, despite a
similar peak flow stimulus. L-NMMA infusion revealed that FMD following wrist occlusion is
mediated exclusively by NO, while dilatation following upper arm occlusion comprises a
substantial component not mediated by NO, most probably related to tissue ischaemia around
the brachial artery. FMD following wrist occlusion may be a more valid marker of endothelial
function than dilatation following upper arm occlusion.
INTRODUCTION
The endothelium plays a key role in the development and
progression of atherosclerosis [1]. Endothelial dysfunction in the coronary arteries of humans is found in the
presence of coronary atherosclerosis and cardiovascular
risk factors [2,3], and precedes the development of
clinically detectable disease [4,5]. In recently published
prospective studies, coronary endothelial dysfunction
was found to be associated with an increased risk of
coronary events independent of classical risk factors
[6,7]. Clear prospective evidence of outcome benefit
Key words: endothelial function, flow-mediated dilatation, nitric oxide, ultrasound.
Abbreviations: EDD, end-diastolic diameter ; FMD, flow-mediated dilatation ; L-NMMA, NG-monomethyl-L-arginine.
Correspondence: Dr J Goodfellow (e-mail GoodfellowJ!cardiff.ac.uk).
# 2001 The Biochemical Society and the Medical Research Society
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following improvement in endothelial function is not yet
available, but considerable circumstantial evidence
supports the strategy of improving endothelial function.
For example, statins, angiotensin-converting enzyme
inhibitors, spironolactone and fish oils improve cardiovascular outcome [8–11], and have also been shown in
separate experimental studies to improve endothelial
function [12–15].
Coronary endothelial function correlates closely with
endothelial function in large peripheral arteries as
measured by flow-mediated dilatation (FMD), indicating
the systemic nature of the abnormality [16]. Furthermore,
there appears to be a graded relationship between
endothelial dysfunction and cardiovascular risk [7], and
FMD of the brachial artery correlates inversely with
the extent of coronary atherosclerosis [17,18].
FMD is a phenomenon whereby increased flow in an
artery results in vasodilatation mediated by changes in
shear stress, detected by endothelial cells. It is abolished
by removal of or damage to the endothelium [19,20]. The
phenomenon is mediated in larger arteries by endothelial
nitric oxide (NO), and selective blockade of NO production with N G-monomethyl-L-arginine (L-NMMA)
abolishes the response [21].
In 1992, Celermajer and colleagues [22] described a
simple yet elegant method to study endothelial function
in humans. They postulated that FMD, measured noninvasively using high-resolution ultrasound, could serve
as an indication of the functional integrity of the
endothelium [22]. FMD assessed by ultrasound measurement of the increase in the diameter of the brachial artery
during reactive hyperaemia is now a widely used noninvasive research tool for the assessment of endothelial
function in humans. However, the technique has not
been standardized and important methodological
differences exist, particularly with regard to the position
of the occlusive cuff relative to the imaged section of the
target vessel. The rationale for using FMD as a surrogate
of NO-mediated endothelial function rests on the assumption that it is an endothelium-dependent, predominantly NO-mediated, process. We sought to compare the NO component of brachial artery dilatation
following release of wrist and upper arm occlusion in
healthy subjects.
METHODS
ture coronary disease (age
60 years), hyperhomocysteinaemia (15 µmol\l) and smoking. A further five
male subjects with the same inclusion\exclusion criteria
were recruited for an L-NMMA dose-ranging study. The
investigations conformed to the principles outlined in
the Declaration of Helsinki, and the Local Research Ethics
Committee approved the study protocol. Written informed consent was obtained from each subject. Baseline
characteristics of the volunteers are shown in Table 1.
Protocol for cuff position study
All volunteers were studied after an 8 h fast. Venous
blood was collected into Vacutainers, and lipids, glucose
and creatinine were analysed conventionally on the day
of sampling. The sample for assay of total plasma
homocysteine was centrifuged immediately [1630 g
(3000 rev.\min) for 10 min] and the plasma was stored at
k70 mC until analysis by enzymic immunoassay (Abbot
IMx ; Abbot Diagnostics). A 27G needle was inserted
into the brachial artery of the non-dominant arm under
local anaesthesia. Normal saline was infused through the
needle at a constant rate of 0.5 ml\min. The brachial
artery was imaged 7–10 cm distal to the puncture site.
After at least 10 min of stabilization, FMD was measured
(see below) following release of a cuff at the wrist (distal
to the ultrasound probe) that was inflated to 250 mmHg
for 5 min (protocol 1). End-diastolic diameter (EDD)
was recorded at 60 s intervals for 5 min, and then at
10 min, to examine the time course of diameter changes
after cuff release. After return of the vessel diameter to a
stable baseline, FMD was measured after release of a cuff
placed on the upper arm (proximal to the ultrasound
probe) that was inflated to 250 mmHg pressure for 5 min
(protocol 2). EDD was recorded at 60 s intervals for
5 min and then at 10 min after cuff release. Following
return of the vessel diameter to baseline, the NO synthase
inhibitor L-NMMA (Clinalfa, La$ nfelfingen, Switzerland)
was infused at 3 mg\min (0.5 ml\min) for 15 min in place
of normal saline. FMD was then measured again using
both protocols during continuous infusion of L-NMMA.
Blood pressure was measured continuously using
photo-plethysmography (Finapres). Blood velocity was
measured using an 8 MHz continuous-wave Doppler
probe mounted in a stereotactic device at an angle of 60m
to the vessel. Blood flow was calculated immediately after
cuff release as the product of the Doppler time velocity
integral, heart rate and brachial artery diameter measured
by ultrasonic wall tracking as described below.
Subjects
For the main study comparing cuff positions, ten healthy
males were recruited. All volunteers were free from
medications and from factors associated with endothelial
dysfunction, namely hyperlipidaemia (total cholesterol
6.5 mmol\l), hypertension (blood pressure 140\
80 mmHg), diabetes mellitus, family history of prema# 2001 The Biochemical Society and the Medical Research Society
Protocol for L-NMMA dose-ranging study
To determine whether complete suppression of stimulated endothelial NO synthase activity was achieved with
L-NMMA after release of the upper cuff, a dose-ranging
study was performed. In five further male subjects an
intra-arterial needle was inserted into the brachial artery
Cuff position determines the NO component of flow-mediated dilatation
Table 1
Characteristics of study subjects
LDL, low-density lipoprotein. Data are expressed as means (S.D.).
Characteristic
Cuff positions study ( n l 10)
L-NMMA
dose-ranging study ( n l 5)
Age (years)
Cholesterol (mmol/l)
Triacylglycerols (mmol/l)
LDL cholesterol (mmol/l)
Glucose (mmol/l)
Homocysteine (µmol/l)
Systolic pressure (mmHg)
Diastolic pressure (mmHg)
Heart rate (beats/min)
34 (5)
5.1 (0.7)
0.88 (0.34)
3.31 (0.7)
5.0 (0.4)
10.2 (1.7)
133 (12)
78 (6)
59 (9)
33 (4)
5.8 (0.8)
1.16 (0.34)
3.92 (0.8)
4.8 (0.1)
9.5 (4.3)
126 (6)
73 (3)
58 (11)
as described above. Dilatation was then measured following release of an upper arm cuff inflated for 5 min
during infusion of : (i) normal saline (control), (ii)
3 mg\min L-NMMA and (iii) 12 mg\min L-NMMA, all
at a constant rate of 0.5 ml\min.
hoc test (Tukey). The time courses of vessel diameter
changes after cuff release were analysed by multivariate
ANOVA. A P value of 0.05 was considered statistically
significant.
Non-invasive measurement of arterial
diameter
RESULTS
FMD was measured using high-resolution ultrasound
and wall tracking, as described previously by us [15].
Using a specially adapted duplex colour flow echo
machine (Toshiba Ultrasound) with a 7.5 MHz linear
phased-array transducer, the brachial artery was imaged
above the antecubital crease, 5–10 cm distal to the
brachial artery puncture site. When a clear B-mode image
of the brachial artery anterior and posterior walls was
obtained, the transducer was fixed by means of a
stereotactic clamp and the position was held constant for
the duration of the study. The radio-frequency signals
from the corresponding M-mode image were relayed to
the wall tracking system (Vadirec, Oosterbeck, The
Netherlands). Following a 10 s acquisition period,
the radio-frequency signal is displayed as a waveform,
allowing manual placement of cursors on the anterior and
posterior brachial artery walls. Vessel wall movements
are then tracked automatically using the acquired data. A
displacement waveform is generated, enabling accurate
measurement of EDD for a series of beats (theoretical
spatial resolution 3 µm) [23]. FMD was reported as the
greatest absolute increase in EDD from baseline (average
of three recordings) during the first 3 min after cuff
release, and is expressed as a percentage of the basal vessel
diameter. The coefficient of variation for the measurement of FMD in our laboratory is 5.6 %.
Dilatation after wrist and upper arm
occlusion under baseline conditions
Statistical analysis
Figure 1 FMD following wrist (lower cuff) and upper arm
(upper cuff) occlusion during control and L-NMMA infusion
(3 mg/min)
Results are expressed as meanspS.E.M. unless otherwise
stated. Dilatation and blood flow data were analysed by
one-way ANOVA, followed when significant by a post-
Dilatation following upper arm occlusion was significantly greater than that observed after wrist occlusion
(11.62p3.17 and 7.25p2.49 % respectively ; P 0.05),
despite there being no difference in the peak flow
stimulus following cuff release at the two positions
(160.9p41 and 155.1p46 ml\min respectively) (Figure
1, Table 2). The difference between the diameter changes
with the two protocols persisted throughout the initial
5 min, with both returning to baseline by 10 min (Figures
2 and 3).
The control infusion was of normal saline.
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Table 2
Baseline and peak flow data during control and L-NMMA infusion in the cuff positions study
The control infusion consisted of normal saline (0.9 % NaCl).
Control
L-NMMA
Cuff
Baseline flow (ml/min)
Peak flow (ml/min)
Lower
Upper
25.3 (10)
23.6 (8)
155.1 (46)
160.9 (41)
P value
0.001
0.001
Baseline flow (ml/min)
Peak flow (ml/min)
15.4 (6)
14.1 (5)
140.2 (37)
157.7 (39)
P value
0.001
0.001
P value*
0.85
0.99
* Comparison of peak hyperaemic flow after cuff release during control compared with L-NMMA infusion.
Figure 2 Time course of vessel diameter changes following
lower (wrist) cuff release during control and L-NMMA infusion
(3 mg/min)
LC, lower cuff. The change in vessel diameter is expressed as a percentage of
baseline diameter. The control infusion was of normal saline.
Figure 3 Time course of vessel diameter changes following
upper (upper arm) cuff release during control and L-NMMA
infusion (3 mg/min)
UC, upper cuff. The change in vessel diameter is expressed as a percentage of
baseline diameter. The control infusion was of normal saline.
Dilatation after wrist and upper arm
occlusion during L-NMMA infusion
During L-NMMA infusion (3 mg\min), dilatation after
wrist occlusion was abolished, while the response following upper arm occlusion was only partially attenu# 2001 The Biochemical Society and the Medical Research Society
Figure 4 Time course of vessel diameter changes following
upper (upper arm) and lower (wrist) cuff release during
L-NMMA infusion (3 mg/min)
UC, upper cuff ; LC, lower cuff ; NS, not significant. The change in vessel diameter
is expressed as a percentage of baseline diameter.
ated. As a result, dilatation differed significantly when
comparing the two cuff positions during L-NMMA
infusion (Figure 1). L-NMMA changed dilatation to a
small overall constriction after release of wrist occlusion
(Figure 2), but only partially decreased the dilatation
following upper arm occlusion (Figure 3). No significant
difference in peak hyperaemic blood flow was observed
between the cuff positions during control or L-NMMA
infusion, indicating no difference in the flow stimulus to
dilatation (Table 2). Comparison of the time courses for
changes in vessel diameter during L-NMMA infusion
after cuff release showed significantly greater dilatation
following upper arm compared with wrist occlusion for
the first 3 min (Figure 4). Heart rate and blood pressure
were unchanged during both study protocols.
L-NMMA dose-ranging study
In the five additional volunteers, dilatation following
upper arm cuff release was 11.88p0.91 % in the control
situation. L-NMMA infusion at 3 mg\min reduced dilatation to 8.31p1.96 % (P l 0.014), with no further
Cuff position determines the NO component of flow-mediated dilatation
Figure 5 Dilatation following upper cuff release during
infusion with normal saline (control), or L-NMMA at 3 or
12 mg/min
NS, not significant.
change during infusion with the higher dose of L-NMMA
(12 mg\min ; 8.46p1.93 % dilatation) (Figure 5). No
differences in blood pressure or heart rate were observed
during the study. Basal brachial artery diameter did not
differ significantly when comparing control with LNMMA (3 mg\min) or L-NMMA (12 mg\min) infusion
(3.85, 3.81 and 3.80 mm respectively).
DISCUSSION
This study has revealed two major findings. First, FMD
following release of upper arm occlusion is significantly
greater than that seen after release of wrist occlusion in
healthy males. This finding is consistent with previous
observations comparing upper arm with forearm occlusion of the same duration [24,25]. Secondly, our data
demonstrate that dilatation after wrist occlusion is
exclusively NO-mediated, while dilatation following
upper arm occlusion is only partially mediated by NO, a
finding not reported previously.
Infusion of the NO synthase inhibitor, L-NMMA,
significantly reduced dilatation with both cuff positions
(Figure 1). However, the response following wrist
occlusion was abolished, while that following upper
arm occlusion was only partially reduced, with significant
differences between the two cuff positions during the
3 min following cuff release (Figure 4). A possible
explanation is that the dose of L-NMMA used
(3 mg\min) was inadequate to block stimulated NO
production following upper arm cuff release. However,
the dose-ranging study found no further suppression of
dilatation using a 4-fold greater dose of L-NMMA,
indicating that the dose chosen was adequate and likely to
be at the top of the dose–response curve.
The greater dilatation observed with upper arm occlusion has been attributed, in part, to a difference in the
flow stimulus observed between cuff positions [25].
However, we found no significant increase in peak flow
after upper arm occlusion, and suggest that a different
flow stimulus does not explain the observed difference in
diameter change. However, other factors are more likely
to explain the observed difference. Upper arm occlusion
is more painful than wrist occlusion [24], presumably
because it causes greater tissue ischaemia. The resulting
ischaemia in the region of the imaged section of the
brachial artery is associated with the release of vasodilatory metabolites (e.g. potassium, adenosine, ATP)
and changes in pH, which could explain the non-NOmediated dilatation observed after upper arm cuff release.
A further possibility is that loss of myogenic tone may
account for greater dilatation after upper arm cuff release.
Brachial artery pressure falls to zero during upper arm
occlusion, but will remain normal during wrist occlusion.
Myogenic influences on diameter are well recognized in
larger conduit vessels [26] such as the brachial artery.
Although hyperaemic flow will rapidly wash out
ischaemic vasodilatory metabolites, restoration of myogenic tone is unlikely to be completed within the first
5 min after cuff release. The profound difference in intraarterial pressures during cuff inflation between the two
protocols will contribute to and possibly account entirely
for the difference in diameter change after cuff
release.
The finding that the wrist cuff protocol results in a
‘ purer ’ assessment of NO activity may not in itself
justify the exclusive use of this technique. Endothelial
dysfunction assessed by wrist cuff occlusion results in
small changes in diameter (dilatation usually 3 %) [15]
that are difficult to measure. A Type-2 error might be the
simple explanation for the inability to identify endothelial
dysfunction in a group of subjects with cardiovascular
risk factors using FMD with forearm occlusion in one
recent study [25]. However, the vast majority of published studies have shown that endothelial dysfunction
can be identified by careful measurement of FMD after
forearm\wrist occlusion, allowing small improvements
following interventions to be detected [15,22,27–31].
The present study had certain limitations. The study
design resulted in measurement of lower cuff and then
upper cuff responses, raising the possibility of an order
effect. However, the results from the L-NMMA doseranging study, where only the upper cuff protocol was
followed (12 mg dose), argue against this possibility, as
no significant differences in the responses following
upper cuff release at baseline were observed between
experiments. Another limitation was the fact that we
measured peak brachial artery flow, but not duration of
peak flow or duration of the hyperaemic response. In
contrast, others have demonstrated a higher peak flow
and longer duration of hyperaemia with upper cuff
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occlusion, and have suggested this as a possible explanation for the greater dilatation seen [32]. The degree
of dilatation produced following cuff occlusion depends
on peak hyperaemic flow and duration of hyperaemia
[33]. L-NMMA does not affect the peak flow observed
following post-ischaemic dilatation, as this is driven by
ischaemic metabolites. L-NMMA will affect the late phase
of the hyperaemic response, which is in part NO
mediated. Therefore it is unlikely that L-NMMA would
have differential effects on the duration of peak flow and
thus influence the FMD observed after upper and lower
cuff occlusion.
In conclusion, the present study has shown that FMD
after release of wrist occlusion represents the activity of
NO. In contrast, dilatation after upper arm occlusion is
greater, with a significant component not mediated by
NO and perhaps not by flow. These differences are not
attributable to differences in the peak flow stimulus, but
are more likely to be the result of ischaemia in the
surrounding tissue or pressure changes in the brachial
artery itself, induced by the occluding cuff. This may
suggest that studies with L-NMMA should be performed
before and after interventions that affect endothelial
function, assessed with an upper arm cuff, as improved
dilatation may not necessarily be due to improved NO
activity.
ACKNOWLEDGMENT
S. N. D. is supported by a Junior Research Fellowship
from the British Heart Foundation.
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Received 22 January 2001/14 May 2001; accepted 18 July 2001
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